Project Summary/Abstract Type 1 Diabetes (T1D) is characterized by a targeted autoimmune attack against self-antigens present on or within the insulin-producing ? cells of the pancreatic islets of Langerhans, resulting in their destruction and subsequent loss of systemic blood glucose homeostasis. Current treatment options include lifelong disease management via frequent blood glucose monitoring and injections with exogenous insulin, but to date, there is no cure or means to prevent the disease in humans. Significant progress has been made improving patients' ability to manage their T1D symptoms (i.e., various forms of insulin, continuous glucose monitoring, and more), but even with rigorous glycemic monitoring, micro- and macro-vascular disease are common and life expectancy reduced. This, combined with high cost of T1D care, supports a pressing need for a safe and effective therapy. Systemic immunosuppression can offer a temporary reprieve from autoimmunity and T1D, but these treatments, associated with toxic side effects, are not antigen specific and thus, do not address the underlying cause of the disease. Previous studies have shown that combining a short course immunosuppressant, anti-CD3 monoclonal antibody (mAb), with daily oral administration of genetically modified Lactococcus lactis (L. lactis) expressing a key T1D autoantigen (proinsulin) and a tolerogenic cytokine (IL-10) (L. lactis PINS-IL-10) prevents and reverses T1D in non-obese diabetic (NOD) mice by inducing lasting antigen-specific immunological tolerance. L. lactis PINS-IL-10 is non-pathogenic, non-colonizing, and lacks the ability to produce the essential amino acid thymidine which serves as a containment system to prevent dissemination into the environment. This innovative L. lactis technology is generally regarded as safe (GRAS) by the FDA and approved for use in humans for the treatment of Crohn?s disease. However, anti-CD3 mAb is not FDA approved for human clinical use. Herein, we propose oral treatment of NOD mice with L. lactis PINS-IL-10 combined with an alternative immunosuppressive agent, anti- thymocyte globulin (ATG), which has been approved for use in humans for decades. We expect that ATG + L. lactis PINS-IL-10 combination therapy will significantly prevent or delay T1D onset in NOD mice. In addition to studies of treatment efficacy, detailed investigations of therapeutic mechanism will be performed including cross-sectional analysis of regulatory T cells, helper T cells, dendritic cells, and innate lymphoid cells by flow cytometry of the gut associated lymphoid tissues (GALT), the target organ (pancreas), and pancreatic draining lymph nodes; functional assays measuring T cell autoantigen recall and regulatory T cell (Treg) suppression; and in vivo adoptive transfer of T1D. Phenotypic or functional changes in the immune cell subsets responsible for oral tolerance induction by the proposed therapy might offer important utility as biomarkers for monitoring therapeutic response in future studies. If effective, this combinatorial treatment can quickly be translated for human use and by modifying the autoantigen secreted by L. lactis, offers potential application in additional autoimmune diseases beyond T1D. Thus, we expect that ATG + L. lactis PINS-IL-10 combination therapy may present a much-needed low-risk intervention to prevent or even cure T1D.